This invention pertains to methods for regulating the healing of damaged tissue by adjusting the concentration of nitric oxide in the vicinity of the damaged tissue.
Nitric oxide (NOxe2x80xa2) is synthesized from the amino acid L-arginine by a family of enzymes termed nitric oxide synthases. Its small size and its unpaired electron (denotedxe2x80xa2), make it a highly reactive and locally diffusible free radical. Since the discovery in 1987 that endothelium derived relaxing factor (EDRF) is, in fact, NOxe2x80xa2, (Palmer, R. M. J. et al., 1988, Nature 333:664-6; Palmer, R. M. J. et al., 1987, Nature, 327:524-527) it has become evident that NOxe2x80xa2 is a widely distributed and multi-functional intra- and intercellular messenger. There is strong evidence that NOxe2x80xa2 synthesized by vascular endothelium is responsible for the regulation of blood pressure and the control of platelet aggregation (Mollace, V. et al., 1990, Biochem. Biophys. Res. Comm., 172:564-9; Rand, M. J. et al., 1992, Clin. Exp. Phamacol. Physiol., 19:147-69; Szabo, C. et al., 1993, Circulation Res., 73:73), and may be involved in vascular injury caused by tissue deposition of immune complexes (Mulligan et al., Proc. Natl. Acad. Sci. USA, 88:6338, 1991). In the central nervous system, nitric oxide is thought to be a neurotransmitter involved in memory and motor function (Bult, H. et al., 1990, Nature, 345:346-347; Dawson, T. M. et al., 1991, Proc. Natl. Acad. Sci. USA, 88:7797-7801; Giovanelli, J. et al., 1991, Proc. Natl. Acad. Sci. USA, 88:7091-7095; Moroz, L. L. et al., 1993, Neuroreport, 4:643-6; Knowles, R. G. et al., 1989, Proc. Natl. Acad. Sci. USA, 86:5159-5162; Snyder, S. H., 1993, Nature, 364:577-632). In the peripheral nervous system, a widespread network of nonadrenergic, noncholinergic nerves use nitric oxide to modulate gastrointestinal, bladder and corpus cavernosum relaxation and erection. (Moncada, S. et al., 1993, New Engl. J. Med., 329 (27):2002-12; Ward, S. M. et al., 1992, Am. J. Physiol, 263:G277-84).
NOxe2x80xa2 is produced in large amounts by an inducible isoform of nitric oxide synthase in macrophages, neutrophils, lymphocytes and peripheral-blood monocytes during immunological reactions and septic shock. (Hevel, J. M. et al., 1991, J. Biol. Chem., 266:22789-22791; Hibbs, J. B. et al, 1988, Biochem. Biophys. Res. Comm., 157:87-94; Nathan, C. F. et al., 1991, Curr. Opin. Immunol., 3:65-70; Salvemini, D. et al., 1990, Biochem. Biophys. Res. Comm., 169:596-601; Szabo, C. et al., 1993, Circulation Res., 73:73). NOxe2x80xa2 is also produced in keratinocytes in response to inflammatory mediators (Reck et al.,J. Biol Chem., 267:21277, 1992.) There is also an inducible form of nitric oxide synthase in cartilage. (Murrell, G. A. C. et al., 1994, International Business Communications 3rd Symposium on Nitric Oxide: Palmer, R. M. et al., 1993, Biochem. Biophys. Res. Comm., 193:398-405; Stadler, J. et al., 1991, J. Immunol, 147:3915-20) Constitutive, Ca++ dependent isoforms of the enzyme found in the brain and blood vessels release NOxe2x80xa2 at low, relatively stable concentrations.
The present invention relates to a previously uncharacterized role for NO in wound healing. Wound healing involves the recruitment of inflammatory cells, followed by fibroblasts, to the site of the wound, where collagen and other connective tissue elements are deposited. The collagen fibers then gradually realign to resemble the original connective tissue (e.g. tendon, ligament, skin.) The ability to regulate this process locally and specifically would be of considerable therapeutic importance e.g. after surgery or trauma. Furthermore, in certain pathological situations, such as arthrofibrosis, Dupuytren""s contracture, peritoneal adhesions, frozen shoulder, scleroderma, and keloid formation, over-expression, and sometimes normal expression, of the repair mechanisms has negative consequences, and it would be desirable to selectively suppress this response.
Conversely, there are many situations in which the healing response in wound healing is delayed or inhibited e.g. in patients with systemic diseases such as liver failure, renal impairment, diabetes, peripheral vascular disease, or in patients taking drugs that inhibit healing e.g. corticosteroids or immunosuppressive agents. In these cases, additional exogenous NOxe2x80xa2 may enhance the healing response.
Therefore, there is a need in the art for methods and compositions to influence wound healing in a temporally and spatially regulatable manner.
It has now unexpectedly been found that NOxe2x80xa2 acts as an early initiator of wound healing in mammals. The present invention encompasses a method for promoting the healing of a damaged tendon or other soft tissue in a patient in need of such treatment, by exposing the tissue or tendon to an increased concentration of NOxe2x80xa2. This increased concentration or level of NOxe2x80xa2 may be achieved by contacting the damaged soft tissue or tendon with an agent that increases the local concentration of NOxe2x80xa2 i.e. the concentration of NOxe2x80xa2 within, or in the immediate vicinity of, the damaged tendon. The agent may be a compound that increases the activity of NO synthase (NOS) within the tendon, or a compound that provides NOxe2x80xa2 as a breakdown product.
In another aspect, the invention encompasses a method for inhibiting unwanted wound healing in a patient in need of such treatment, as in e.g. arthrofibrosis, Dupuytren""s contracture, peritoneal adhesions, frozen shoulder, scleroderma, or keloid formation, by contacting the affected tissue with an agent that inhibits the activity of nitric oxide synthase in the tissue.
In another aspect, the invention provides pharmaceutical compositions for promoting the healing of a damaged tissue, comprising an agent that increases the concentration or level of nitric oxide in the vicinity of the damaged tissue. The invention also provides pharmaceutical compositions for inhibiting unwanted wound healing in or near a damaged tissue, comprising an agent that reduces the concentration or level of nitric oxide in the vicinity of the damaged tissue.
In still another aspect, the invention provides a method for identifying agents that stimulate the production of nitric oxide in damaged tendons, which comprises the steps of:
(i) administering a test compound to a mammal;
(ii) damaging the Achilles tendon in the mammal;
(iii) allowing the damaged tendon to heal for a predetermined time;
(iv) measuring nitric oxide synthase activity in the tendon; and
(v) comparing the nitric oxide synthase activity with the nitric oxide synthase activity in an untreated, damaged tendon to determine that the compound causes a significant increase in nitric oxide synthase activity over that observed in a control tendon.
In still another aspect, the invention provides a method for identifying agents that inhibit unwanted wound healing, which comprises the steps of:
(i) administering a test compound to a mammal;
(ii) damaging the Achilles tendon in the mammal;
(iii) allowing the damaged tendon to heal for a predetermined time;
(iv) measuring the activity of nitric oxide synthase in the tendon; and
(v) comparing the nitric oxide synthase activity with the nitric oxide synthase activity in an untreated damaged tendon to determine that the compound causes a significant decrease in nitric oxide synthase activity over that observed in a control tendon.